Introgression of Brown Norway \u3cem\u3eCYP4A\u3c/em\u3e Genes onto the Dahl Salt-Sensitive Background Restores Vascular Function in SS-5\u3csup\u3eBN\u3c/sup\u3e Consomic Rats

The present study tested the hypothesis that the Dahl SS (salt-sensitive) rat has vascular dysfunction due, in part, to the up-regulation of the CYP4A/20-HETE (cytochrome P450 ω-hydroxylase 4A)/20-hydroxyeicosatetraenoic acid) system. To assess the role of vascular 20-HETE, SS rats were compared with SS-5BN consomic rats, carrying CYP4A alleles on chromosome 5 from the normotensive BN (Brown Norway) introgressed on to the SS genetic background. Cerebral arteries from SS-5BN rats had less CYP4A protein than arteries from SS rats fed either NS (normal-salt, 0.4% NaCl) or HS (high-salt, 4.0% NaCl) diet. ACh (acetylcholine)-induced dilation of MCAs (middle cerebral arteries) from SS and SS-5BN rats was present in SS-5BN rats fed on either an NS or HS diet, but absent in SS rats. In SS rats fed on either diet, ACh-induced dilation was restored by acute treatment with the CYP4A inhibitor DDMS (N-methyl-sulfonyl-12,12-dibromododec-11-enamide) or the 20-HETE antagonist 20-HEDE [20-hydroxyeicosa-6(Z),15(Z)-dienoic acid]. The restored response to ACh in DDMS-treated SS rats was inhibited by L-NAME (NGnitro-L-arginine methyl ester) and unaffected by indomethacin or MS-PPOH [N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide]. Vascular relaxation responses to the NO donor C5FeN6Na2O were intact in both SS and SS-5BN rats and unaffected by the acute addition of DDMS, indicating that the vascular dysfunction of the SS rat is due to a reduced bioavailability of NO instead of failure of the VSMCs (vascular smooth muscle cells) to respond to the vasodilator. Superoxide levels in cerebral arteries of SS-5BN rats [evaluated semi-quantitatively by DHE (dihydroethidium) fluorescence] were lower than those in the arteries of SS rats. These findings indicate that SS rats have an up-regulation of the CYP4A/20-HETE pathway resulting in elevated ROS (reactive oxygen species) and reduced NO bioavailability causing vascular dysfunction

Mannose-6-phosphate regulates destruction of lipid-linked oligosaccharides.

Mannose-6-phosphate (M6P) is an essential precursor for mannosyl glycoconjugates, including lipid-linked oligosaccharides (LLO; glucose(3)mannose(9)GlcNAc(2)-P-P-dolichol) used for protein N-glycosylation. In permeabilized mammalian cells, M6P also causes specific LLO cleavage. However, the context and purpose of this paradoxical reaction are unknown. In this study, we used intact mouse embryonic fibroblasts to show that endoplasmic reticulum (ER) stress elevates M6P concentrations, leading to cleavage of the LLO pyrophosphate linkage with recovery of its lipid and lumenal glycan components. We demonstrate that this M6P originates from glycogen, with glycogenolysis activated by the kinase domain of the stress sensor IRE1-α. The apparent futility of M6P causing destruction of its LLO product was resolved by experiments with another stress sensor, PKR-like ER kinase (PERK), which attenuates translation. PERK's reduction of N-glycoprotein synthesis (which consumes LLOs) stabilized steady-state LLO levels despite continuous LLO destruction. However, infection with herpes simplex virus 1, an N-glycoprotein-bearing pathogen that impairs PERK signaling, not only caused LLO destruction but depleted LLO levels as well. In conclusion, the common metabolite M6P is also part of a novel mammalian stress-signaling pathway, responding to viral stress by depleting host LLOs required for N-glycosylation of virus-associated polypeptides. Apparently conserved throughout evolution, LLO destruction may be a response to a variety of environmental stresses.This work is supported by NIH grants DK-042394, HL-052173, and HL-057346 to R.J.K.; by NIH grants AI-073898 and GM-056927 to I.M.; by NIH grant R-37-DK047119 and a Principal Research Fellowship from the Wellcome Trust to D.R.; by NIH grant GM-031278 and support from the Robert Welch Foundation to J.R.F.; and by NIH grant GM-038545 and Robert Welch Foundation grant I-1168 to M.A.L

Development of Epoxyeicosatrienoic Acid Analogs with in Vivo Anti-Hypertensive Actions

Epoxyeicosatrienoic acids (EETs) contribute importantly to the regulation of vascular tone and blood pressure control. The purpose of this study was to develop stable EET analogs and test their in vivo blood pressure lowering effects in hypertensive rats. Using the pharmacophoric moiety of EETs, ether EET analogs were designed with improved solubility and resistance to auto-oxidation and metabolism by soluble epoxide hydrolase. Ether EET analogs were chosen based on their ability to dilate afferent arterioles and subsequently tested for blood pressure lowering effects in rodent models of hypertension. Initially, 11,12-ether-EET-8-ZE failed to lower blood pressure in angiotensin hypertension or spontaneously hypertensive rats (SHR). Esterification of the carboxylic group of 11,12-ether-EET-8-ZE prevented blood pressure increase in SHR when injected at 2 mg/day for 12 days (MAP Δ change at day 8 of injection was −0.3 ± 2 for treated and 12 ± 1 mmHg for control SHR). Amidation of the carboxylic group with aspartic acid produced another EET analog (NUDSA) with a blood pressure lowering effect when injected at 3 mg/day in SHR for 5 days. Amidation of the carboxylic group with lysine amino acid produced another analog with minimal blood pressure lowering effect. These data suggest that esterification of the carboxylic group of 11,12-ether-EET-8-ZE produced the most effective ether-EET analog in lowering blood pressure in SHR and provide the first evidence to support the use of EET analogs in treatment of cardiovascular diseases

19-Hydroxyeicosatetraenoic Acid Analogs: Antagonism of 20-Hydroxyeicosatetraenoic Acid-Induced Vascular Sensitization and Hypertension

19-Hydroxyeicosatetraenoic acid (19-HETE, 1), a metabolically and chemically labile cytochrome P450 eicosanoid, has diverse biological activities including antagonism of the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE, 2). A SAR study was conducted to develop robust analogs of 1 with improved in vitro and in vivo efficacy. Analogs were screened in vitro for inhibition of 20-HETE-induced sensitization of rat renal preglomerular microvessels toward phenylephrine and demonstrated to normalize the blood pressure of male Cyp4a14(-/-) mice that display androgen-driven, 20-HETE-dependent hypertension

Epoxyeicosatrienoic Acid Agonist Regulates Human Mesenchymal Stem Cell–Derived Adipocytes Through Activation of HO-1-pAKT Signaling and a Decrease in PPARγ

Human mesenchymal stem cells (MSCs) expressed substantial levels of CYP2J2, a major CYP450 involved in epoxyeicosatrienoic acid (EET) formation. MSCs synthesized significant levels of EETs (65.8 ± 5.8 pg/mg protein) and dihydroxyeicosatrienoic acids (DHETs) (15.83 ± 1.62 pg/mg protein), suggesting the presence of soluble epoxide hydrolase (sEH). The addition of an sEH inhibitor to MSC culture decreased adipogenesis. EETs decreased MSC-derived adipocytes in a concentration-dependent manner, 8,9- and 14,15-EET having the maximum reductive effect on adipogenesis. We examined the effect of 12-(3-hexylureido)dodec-8(Z)-enoic acid, an EET agonist, on MSC-derived adipocytes and demonstrated an increased number of healthy small adipocytes, attenuated fatty acid synthase (FAS) levels (P < 0.01), and reduced PPARγ, C/EBPα, FAS, and lipid accumulation (P < 0.05). These effects were accompanied by increased levels of heme oxygenase (HO)-1 and adiponectin (P < 0.05), and increased glucose uptake (P < 0.05). Inhibition of HO activity or AKT by tin mesoporphyrin (SnMP) and LY2940002, respectively, reversed EET-induced inhibition of adipogenesis, suggesting that activation of the HO-1-adiponectin axis underlies EET effect in MSCs. These findings indicate that EETs decrease MSC-derived adipocyte stem cell differentiation by upregulation of HO-1-adiponectin-AKT signaling and play essential roles in the regulation of adipocyte differentiation by inhibiting PPARγ, C/EBPα, and FAS and in stem cell development. These novel observations highlight the seminal role of arachidonic acid metabolism in MSCs and suggest that an EET agonist may have potential therapeutic use in the treatment of dyslipidemia, diabetes, and the metabolic syndrome

14,15-Epoxyeicosa-5,8,11-trienoic Acid (14,15-EET) Surrogates: Carboxylate Modifications

The cytochrome P450 eicosanoid 14,15-epoxyeicosa-5,8,11-trienoic acid (14,15-EET) is a powerful endogenous autacoid that has been ascribed an impressive array of physiologic functions including regulation of blood pressure. Because 14,15-EET is chemically and metabolically labile, structurally related surrogates containing epoxide bioisosteres were introduced and have become useful in vitro pharmacologic tools but are not suitable for in vivo applications. A new generation of EET mimics incorporating modifications to the carboxylate were prepared and evaluated for vasorelaxation and inhibition of soluble epoxide hydrolase (sEH). Tetrazole <b>19</b> (ED₅₀ 0.18 μM) and oxadiazole-5-thione <b>25</b> (ED₅₀ 0.36 μM) were 12- and 6-fold more potent, respectively, than 14,15-EET as vasorelaxants; on the other hand, their ability to block sEH differed substantially, i.e., 11 vs >500 nM. These data will expedite the development of potent and specific in vivo drug candidates